Malaria is the most important human parasitic disease. More than forty percent of the world's population live in areas where malaria is transmitted (e.g., parts of Africa, Asia, the Middle East, Central and South America, Hispaniola, and Oceania). An estimated 700,000-2.7 million persons die of malaria each year, 75% of them being African children.
Biochemical and genetic analyses have shown that proteases of Plasmodium, the causative agent of malaria, play a central role in the entrance of the sporozoite and the merozoite into the host hepatocyte or red blood cell (RBC), respectively. The surface proteins of both extracellular invasive forms undergo obligatory proteolytic processing executed by parasite-encoded serine proteases, which are thus directly accessible to host factors such as antibodies or drugs. Importantly, 60% of the plasmatic proteins are protease inhibitors (mainly involved in the regulation of coagulation or complement activation) suggesting that the parasitic proteases active on the outer surface of the parasite are highly specific, differ from the host proteases and are insentive to host plasmatic protease inhibitors. Altogether, the features of the parasite serine proteases involved in RBC and hepatocytes invasion make them attractive targets as novel anti-malarials.
SUB2 and SUB1 are two essential Plasmodium serine proteases which are known to be involved in host cells invasion. The SUB2 subtilisin-like serine protease is discharged by the parasite onto the surface of the extracellular merozoite, where it performs proteolytic processing of major parasite surface proteins, a final maturation step that is essential for host cell invasion. SUB2 sequence is highly conserved in P. falciparum and P. vivax. Because of all its interesting properties, SUB2 has been described as a novel anti-malarial drug target in International PCT patent application WO2006/120579. The SUB1 enzyme has been shown to be involved in the egress of Plasmodium from infected erythrocytes and plays also a yet undefined role during the RBC invasion process per se. The SUB1 enzyme of P. falciparum has also been the subject of a fluorescence-based assay for identifying inhibitors of P. falciparum (Blackman et al. (2002), Biochemistry, 41, 12244-12252). SUB2 and SUB1 share substantial inter-species structural homology in their catalytic domains (e.g. >75% sequence identity between the PfSUB2 and PvSUB2 domains, and between PfSUB1 and PvSUB1 domains). The Plasmodium genome harbours a third prokaryotic subtilisin-like serine protease, SUB3, which differs from SUB1 and SUB2 in being not essential for the intra-erythrocytic cycle. However, its expression is activated after the entry of the sporozoites into the hepatocytes, suggesting a role during the establishment of the infectious process in mammalian hosts.
Chloroquine is a 4-aminoquinoline drug used in the treatment or prevention of malaria. Popular drugs based on chloroquine phosphate (also called nivaquine) are Chloroquine FNA, Resochin and Dawaquin. Worryingly, resistance to both Plasmodium falciparum and P. vivax, the two main species infecting humans, have eroded treatment efficacy and malaria control measures. In addition, mosquito resistance to insecticides is spreading. Efforts at developing a malaria vaccine with long term efficiency have met with limited success.
There is thus an urgent need for the discovery, screening and development of novel anti-malarials. There is also a need for compounds targeting Plasmodium invasion process of either the hepatocyte or the red blood cells. There is also a need for enzyme inhibitors effective for prophylaxis preventing host infection.